Borna disease virus-specific protein and kits

ABSTRACT

Borna disease virus (BDV) causes a rare neurological disease in horses and sheep. A subtractive cDNA expression library was constructed with poly A-selected RNA from a BDV infected MDCK cell line. A clone (B8) was isolated that specifically hybridizes to RNA isolated from BDV-infected brain tissue and BDV-infected cell lines. This clone hybridizes to four BDV-specific positive strand RNAs and one negative strand RNA in BDV-infected rat brain. Nucleotide sequence analysis of the clone suggests that it represents a full length mRNA which contains several open reading frames. The Borna Disease Virus DNA sequences as well as proteins encoded by the BDV DNA sequences are provided.

This invention was made using U.S. government funds awarded by theNational Institutes of Health. Therefore the government retains certainrights in the invention.

This is a continuation of application Ser. No. 07/618,288 filed Nov. 28,1990, now abandoned.

BACKGROUND OF THE INVENTION

Borna disease is an infectious neurological disease that occurssporadically in horses and sheep in Central Europe (Ludwig, Prog. Med.Virol. 35:107 (1988)). Brain homogenates from infected animals can beused to infect a large number of animal species from rodents tonon-human primates (Carbone, Virol. 61:3431 (1987); Narayan, Science220:1401 (1983); Sprankel, Med. Microbiol. Innumol. 165:1 (1978)).Studies in rats have shown that the agent is highly neurovirulent andinvades the brain from peripheral sites by axonal transport (Carbone,supra). It replicates in specific groups of neurons in the cerebralcortex and causes biphasic behavioral disease characterized byaggression and hyperactivity during the acute phase of infection andapathy and eating disorders during the chronic stage (Narayan, supra).In tree shrews (Turpaia glis), infection is associated with disruptionin social interactions (Sprankel, supra). In addition, recent studiesdemonstrate the presence of anti-Borna Disease Virus (BDV) antibodies inhumans with psychiatric illnesses which include personality disordersand schizophrenia (Rott, Science, 228:755 (1985); Amsterdam, Arch. Gen.Psychiatry, 42:1093 (1985)).

The BDV virus replicates in cell cultures and rat brain with thesynthesis of novel 38/39, 24 and 14 kD proteins (Haas, J. Gen. Virol.67:235 (1986)). However, the virus has not been classified since neithera particle nor a specific nucleic acid has been identified in infectiousmaterial.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a DNA molecule which isspecific for the Borna Disease Virus.

It is yet another object of the invention to provide Borna Disease Virusproteins which are substantially pure.

It is an object of the invention to provide means to isolate BornaDisease Virus-related human viruses.

It is still another object of the invention to provide kits and methodsfor diagnosing Borna Disease Virus infections and Borna DiseaseVirus-related human virus infections in humans.

These and other objects of the invention are provided by one or more ofthe embodiments which are described below. In one embodiment, a DNAmolecule is provided which is complementary to the genome of BDV.

In another embodiment a DNA molecule is provided which is complementaryto BDV-encoded messenger RNA.

In still another embodiment of the invention a duplex DNA molecule isprovided which comprises a DNA molecule which is complementary to thegenome of Borna Disease Virus or a DNA molecule which is complementaryto a Borna Disease Virus-encoded messenger RNA.

In yet another embodiment of the invention a preparation of BornaDisease Virus-encoded protein is provided, which preparation issubstantially free of mammalian proteins.

In another embodiment of the invention a DNA molecule is provided whichis complementary to a BDV-related, human virus-encoded messenger RNA.

In still another embodiment of the invention a DNA molecule is providedwhich has the same polarity as a BDV-related, human virus-encodedmessenger RNA, said molecule hybridizing to a DNA molecule which iscomplementary to a Borna Disease Virus-encoded messenger RNA.

In yet another embodiment of the invention a duplex DNA molecule isprovided which comprises a first DNA molecule which is complementary toa BDV-related, human virus-encoded messenger RNA or a second DNAmolecule having the same polarity as a BDV-related, human virus-encodedmessenger RNA, said second DNA molecule hybridizing to a DNA moleculewhich is complementary to a Borna Disease Virus-encoded messenger RNA.

In another embodiment of the invention a method is provided fordetermining a Borna Disease Virus infection in animals or a BornaDisease Virus-related human virus infection in humans, comprising thesteps of:

providing a serum sample of an animal or human to be tested:

contacting the serum sample with a preparation of Borna DiseaseVirus-encoded protein substantially free of mammalian proteins, underconditions where antibody-antigen complexes form and are stable;

determining the presence of antibody-antigen complexes which containsaid Borna Disease Virus-encoded protein, the presence ofantibody-antigen complexes which contain said Borna DiseaseVirus-encoded protein indicating a Borna Disease Virus infection or aBorna Disease Virus-related human virus infection.

In yet another embodiment of the invention a kit is provided fordetermining a Borna Disease Virus infection in animals or a BornaDisease Virus-related human virus infection in humans, comprising:

a solid support; and

a preparation of Borna Disease Virus-encoded protein substantially freeof mammalian proteins, said Borna Disease Virus-encoded proteins boundto said solid support.

In another embodiment of the invention a method is provided fordetermining a Borna Disease Virus infection in animals or a BornaDisease Virus-related human virus infection in humans, comprising thesteps of:

providing a sample of cells from an animal or human to be tested;

extracting RNA from said cells;

contacting said RNA with a DNA probe, said probe comprising a DNAmolecule which is complementary to a Borna Disease Virus-encodedmessenger RNA;

determining the presence of hybrid molecules comprising said probe, thepresence of said hybrid molecules indicating a Borna Disease Virusinfection or a Borna Disease Virus-related human virus infection.

In still another embodiment of the invention a method is provided fordetermining a Borna Disease Virus infection in animals or a BornaDisease Virus-related human virus infection in humans, comprising thesteps of:

providing a sample of cells from an animal or human to be tested;

permeabilizing said cells;

contacting said permeabilized cells with a DNA probe, said probecomprising a DNA molecule which is complementary to a Borna DiseaseVirus-encoded messenger RNA;

determining the presence of hybrid molecules comprising said probe, thepresence of said hybrid molecules indicating a Borna Disease Virusinfection or a Borna Disease Virus-related human virus infection.

In another embodiment of the invention a method is provided fordetermining a Borna Disease Virus-related human virus infection inhumans, comprising the steps of:

providing a sample of cells from a human to be tested;

extracting RNA from said cells;

contacting said RNA with a DNA probe, said probe comprising a DNAmolecule which is complementary to a Borna Disease Virus-related humanvirus-encoded messenger RNA;

determining the presence of hybrid molecules comprising said probe, thepresence of said hybrid molecules indicating a Borna DiseaseVirus-related human virus infection.

In still another embodiment of the invention a method is provided fordetermining a Borna Disease Virus-related human virus infection inhumans, comprising the steps of:

providing a sample of cells from a human to be tested;

permeabilizing said cells;

contacting said permeabilized cells with a DNA probe, said probecomprising a DNA molecule which is complementary to a Borna DiseaseVirus-related human virus-encoded messenger RNA;

determining the presence of hybrid molecules comprising said probe, thepresence of said hybrid molecules indicating a Borna DiseaseVirus-related human virus infection. These and other embodiments of theinvention provide the art with nucleic acids and proteins which can beused to diagnose BDV and BDV-like infections of humans and othermammals. In addition, it provides the tools for isolation andidentification of the viral agents which cause these neurologicaldiseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows specific hybridization of a cDNA clone to RNA isolated fromBDV-infected cells and rat brain.

Northern blot analysis of RNA (10 ug/lane) from: (a) uninfected MDCK(Madin Darby canine kidney) cells; (b) BDV-infected MDCK cells; (c)uninfected rat brain; (d) BDV-infected rat brain; (e) BDV-infected MDCKcells; and (f) BDV-infected rat brain. The northern blot was hybridizedwith a nick translated probe made from DNA from the B8 clone.

FIG. 2 shows the determination of polarity of BDV-specific RNAs

A. Negative strand ³² P-UTP-labeled RNA transcripts of clone B8hybridized to Northern blots containing: (a) 7.5 ug of RNA fromuninfected rat brain; (b) 7.5 ug of RNA from BDV-infected rat brain; and(c) 2.0 ug of poly A selected RNA from BDV-infected rat brain.

B. Positive strand ³² P-UTP-labeled RNA transcripts of clone P4hybridized to northern blots containing: (a) 7.5 ug of RNA fromuninfected rat brain; and (b) 7.5 ug of RNA from BDV-infected rat brain.RNA from BDV-infected rat brain was heated at 90° C. for 2 min andchromatographed on oligo-dt cellulose and RNA was used in northernblots: (c) 5 ug of total RNA from BDV-infected rat brain; (d) 3 ug RNAfrom oligo-dt column wash; and (e) 3 ug of Poly A-containing RNA.

FIGS. 3A and 3B shows the results of in situ hybridization of BDVinfected rat brain with the B8 clone. Brains from (a) uninfected and (b)BDV-infected rats were fixed in formaldehyde, dehydrated andparaffin-embedded.

FIG. 4 shows the identification of BDV-specific proteins encoded by theB8 clone. Poly-A selected RNA from uninfected rat brain (lanes a and b)and BDV-infected rat brain (lanes c, d,l, m and n) were translated invitro and immunoprecipitated with polyclonal anti-BDV sera from rat(lanes a,c and 1), rabbit (lanes b and d), or monoclonal antibodies tothe 38/39 kD protein (lane m) and the 24 kD protein (lane n). In vitrotranscription with T7 polymerase synthesized the negative stranded RNAfrom the 38 clone and positive stranded RNA from the P4 clone. RNAtranscribed in vitro from clone B8 (lanes e and f) and clone P4 (lanesg, h, o and p) were translated in vitro and immunoprecipitated withpolyclonal anti-BDV sera from rat (lanes e and g) rabbit (lanes f and h)or monoclonal antibodies to the 38/39 kD protein (lane o) and 24 kDprotein (lane p). Controls (lanes i, j, q and r) are in vitrotranslation reactions without RNA which were immunoprecipitated withpolyclonal anti-BDV sera from rats (lane i), rabbit (lane j) 38/39 kDmonoclonal antibodies (lane q) and 24 kD monoclonal antibodies (lane r).Molecular weight markers (¹⁴ C-labelled) in lane k contain from top tobottom 200, 92.5, 69, 46, 30, 21.5, and 14.3 kD proteins.

FIG. 5 shows analyses of antibodies in human serum byimmunoprecipitation of the BDV-specific proteins translated from the B8clone.

The lanes contain 1×10⁵ cpm of ³⁵ S-methionine-labelled proteinsimmunoprecipitated with the following sera: BDV infected rat (a); normalrat (b); human anti-BDV-p24 (c); human anti-BDV-p38/39(d); patient 112(e); patient 114 (f); patient 115 (g), and normal human sera in lanes(h-k). Lane M contains molecular weight markers (¹⁴ C-labelled) from topto bottom 200, 92.5, 69, 46, 30, 21.5 and 14.3 kD proteins.

DETAILED DESCRIPTION OF THE INVENTION

Isolation of BDV-specific cDNA

To identify and clone a BDV-specific nucleic acid a subtractive cDNAlibrary was constructed from the mRNA isolated from uninfected andBDV-infected MDCK cell lines. Two rounds of selection were performed toenrich the cDNA library for BDV-specific cDNAS. The vector used forcloning contained a polylinker in the lac z gene into which inserts werecloned; this allowed bacterial expression of a fusion protein. Colonieswere grown in the presence of IPTG to induce expression from the lac zpromoter and were screened using monoclonal antibodies specific for theBDV 38/39 kD protein (Haas, J. Gen Virol. 67:235 (1986)). Nine antibodypositive clones were identified. The DNA from these clones was nicktranslated and hybridized to Northern blots containing RNA fromuninfected and BDV-infected rat brain. One clone specifically hybridizedto RNA from infected rat brain and showed no hybridization to RNA fromuninfected rat brain (FIG. 1). This clone, called B8, contained a 700 bpBDV-specific insert which is an apparently full length mRNA. The B8clone hybridized to four RNAs of 10.5 or 8.7, 3.6, 2.1 and 0.85 kb inBDV-infected rat brain and cell cultures (FIG. 1). These four RNAs areenriched by poly A selection and are apparently positive strand mRNAs.Further, it was reproducibly found that the largest RNA species in ratbrain was 10.5 kb in contrast to 8.7 kb in BDV-infected cell cultures(FIG. 1, lanes e and f). This difference may reflect the presence of adefective genome in the persistently infected cell cultures.

Methods: Standard molecular techniques were used (Maniatis, MolecularCloning, Cold Spring Harbor Laboratory, (1990)). The cDNA libraries wereconstructed using poly A selected RNA from uninfected and infected MDCKcells using reagents and methods in the Invitrogen Librarian kit. Themodified Gubler Hoffman technique (Gubler, Gene 25:263 (1983); Duguid,P.N.A.S. 85:5738 (1988); Sive, Nuc. Acids Res. 16:10937 (1988); Dower,Nuc. Acids Res 16:6127 (1988)) was used for the synthesis of the cDNA;cDNAs were not size selected. Colonies of the subtracted library weregrown in the presence of IPTG and lifted onto nylon membranes. Themicrowave method was used to fix proteins to the filters (Invitrogen). Amix of monoclonal antibodies to the 38/39 kD Borna-specific antigen wasused to detect clones expressing Borna-specific proteins. The DNA fromnine positive clones was nick translated and used as probes on northernblots of RNA from BDV-infected and uninfected rat brain. RNA wasprepared using standard techniques (Chomczynski, Analytical Biochem.162:156 (1987); Puissant, Biotechniques 8:148 (1990)). Samples were runon a formaldehyde denaturing gel and transferred to nylon membranes(Thomas, P.N.A.S. 77:5201 (1980)). Sizes were determined by comparisonwith a radiolabeled RNA ladder (BRL). Specific hydridization to the RNAfrom infected brain was detected in one of the nine clones (clone B8).

Polarity of RNA Species

To determine the polarity of the RNA species in the BDV-infected cellcultures and tissues, strand specific RNA probes were made from the B8clone. The polarity of the B8 clone was determined by sequence analysis.The sequence of the B8 clone is provided in SEQ ID NO: 1. At one end ofthe clone a stretch of As were found and at the other end a number ofATGs were found preceding open reading frames. The negative strand probeshowed a similar hybridization pattern to the nick translated B8 cloneand identified four BDV-specific transcripts (10.5, 3.6, 2.1 and 0.85kb) in BDV infected rat brain RNA (FIG. 2A). This negative strand probealso hybridized to these same sized RNAs from poly A selected RNA (FIG.2A). The presence of apparently subgenomic mRNAs (3.6, 2.1 and 0.85 kb)all of which hybridize with the negative strand of the B8 clone, suggestthat these mRNAs contain common sequences. In contrast, the positivestrand probe hybridized only to a 10.5 kb RNA in BDV-infected rat brain(FIG. 2B).

Methods: Clone P4 was constructed by subcloning the entire B8 cDNAinsert into the PGEM3 vector (Promega). Sequence analysis revealed thatT7 polymerase would direct transcription of the positive strand of theP4 cDNA clone and the negative strand of the B8 cDNA clone. Afterlinearizing the plasmids with restriction enzyme digestion, T7polymerase was used to direct RNA synthesis in vitro for bothconstructs, ² P-UTP was used in the reaction to label the transcripts.RNA was isolated from tissues and cells and Northern blots were preparedas described in FIG. 1. Polyadenylated RNA was selected bychromatography on oligo-dt cellulose. Hybridization to Northern blotswas done using standard conditions. Stringent washes were performedusing 0.1×SSC at 65° C. for 2 hours.

No BDV Sequences in Genomic DNA

In order to eliminate the possibility that the BDV agent is a DNA virusor that the B8 clone represents a cellular gene whose expression wasincreased in BDV-infected cells, Southern hybridization was done withDNA isolated from uninfected and BDV-infected rat brain and MDCK cells.A single copy gene equivalent of the B8 clone was run in parallel andwas detected under the hybridization conditions used (Southern, J. Mol.Biol. 98:503 (1975); Hogan, in Manipulating the Mouse Embryo, ColdSpring Harbor Laboratory, 180 (1986)). Using the nick translated B8clone as a probe (Rigby, J. Mol. Biol. 113:237 (1977)), no hybridizationwas detected on Southern blots containing 10 ug of DNA from either theBDV-infected or uninfected tissues and cells (data not shown).

In Situ Hybridization

In order to further demonstrate the specificity of the B8 clone for theBDV agent, the clone was labelled with ³⁵ S-ATP by nick translation andin situ hybridization was done on sections of uninfected andBDV-infected rat brain. The B8 clone hybridized only to RNA in infectedbrain. Hybridization was localized to neurons in the cerebral cortex,thalamus, the dentate gyrus and the CA4 and CA3 regions of the pyramidalgyrus of the hippocampus (FIG. 3b). This pattern of hybridization isremarkably similar to the localization of BDV antigen established byclassical histopathological and immunocytochemical techniques(Gosztonyi, Neuropsychiatry Clinics, 3:107 (1984)).

Methods: Six uM tissue sections were deparaffinized and treated aspreviously described (Zink, J. Pathol. 136:1250 (1990)). The DNA fromthe B8 clone was radiolabelled by nick translation using [³⁵ S]dATP and[³⁵ S]dCTP (Rigby, supra; Zink supra,) as previously described (Thomas,supra). Specific activities of the radiolabelled DNA probes vere greaterthan 7×10⁸ cpm/ug. Radiolabelled DNA at a concentration of 0.2 ug/ml washybridized to the sections as previously described (Zink, supra). Theslides were exposed for 2 to 5 days, developed and examined by lightmicroscopy; the presence of viral RNA was indicated by silver grainsover cells. Sections of uninfected and BDV-infected rat brain hybridizedwith an irrelevant probe were used as negative controls for everyhybridization reaction. To determine whether the B8 clone encodedBDV-specific proteins, the clone was transcribed in vitro in bothorientations. The RNAs were translated in a rabbit reticulocyte lysatewith ³⁵ S-methionine and the proteins analyzed by immunoprecipitationwith polyclonal and monoclonal antibodies to BDV (FIG. 4). In parallel,poly A selected RNA from BDV-infected rat brain was translated andanalyzed by immunoprecipitation with the same BDV-specific antibodies(FIG. 4). RNA corresponding to the positive strand of the B8 clonedirected the synthesis of four proteins (24, 15.5, 14.5 and 13 kD) whichwere recognized by BDV-specific polyclonal antibodies. In addition, somemonoclonal antibodies to both the 38/39 kD and 24 kD BDV proteinsimmunoprecipitated the same bands (kindly provided by L. Stitz). ThemRNA from rat brain directed the synthesis of the 8/39, 24, 15.5 14.5and 13 kD proteins which were immunoprecipitated by the BDV-specificpolyclonal and monoclonal antibodies. These data provide furtherevidence that the B8 clone is a BDV-specific nucleic acid and encodesBDV-specific proteins.

Methods: Rat brain RNA was isolated and poly A selected (Maniatis,supra, Chomczynski, supra, and Puissant, supra). 0.5 ug of poly Aselected RNA was translated in vitro using Staphylococcalnutleasetreated rabbit reticulocyte lysate (Promega) and ³⁵ S-methionine as alabel. In vitro transcription of clones B8 and P4 was carried out with 1ug of linearized DNA template in a standard reaction with T4 polymeraseand m'G(5') pppG (cap analog) (Stratagene). 0.1 ug of this RNA was usedin the in vitro translation system described above. Immunoprecipitation(using 1×10⁵ cpm/sample) was carried out at 37° C. for one hour(polyclonal sera) or 4 hours (monoclonal sera) followed by addition ofProtein A Sepharose beads and overnight incubation at 4° C.(14). Sampleswere run on 5 to 20% gradient SDS polyacrylamide gels and fluorographywas performed (Entensify, New England Nuclear). Gels were dried andexposed at -70° C.

Immunoprecipitation of BDV Proteins by Human Sera

Antibodies to BDV have previously been identified in the serum ofpsychiatric patients by indirect immunofluorescence (Rott, supra; andAmsterdam, supra). To examine whether anti-BDV antibodies in human serawould recognize the virus-specific proteins encoded by the B8 clone,sera from normal controls (Rott, supra) and psychiatric patients (Rott,supra) with behavioral disorders from areas in Germany endemic for BDVwere tested. The serum from two of the patients had previously beenshown to have antibodies to BDV infected MDCK cells by indirectimmunofluorescence. One serum recognized the BDV 24 kD and one the B8 kDprotein on Western blots using homogenates of BDV-infected rat brain (S.Herzog, unpublished observations). To determine if antibodies in thesehuman sera would recognize the 24 kD protein encoded by the B8 clone,35S labeled protein translated in vitro (as described above) wasanalyzed by immunoprecipitation. Antibodies in three out of seven of thepatients' sera recognized the 24 kD protein (FIG. 5), including theserum previously shown by Western blot to recognize the 24 kD protein.It has been observed by Western blot analysis of human sera that eitherthe 24 kD or the 38/39 kD protein is recognized but not both which ischaracteristic of immune serum from animals infected with BDV. However,no antibodies to the BDV protein were detected in seven normal controls.

These results suggest that a BDV-related virus is involved in behavioraldisease in humans. Further, this cross reactivity will allow the use ofcloned BDV proteins and nucleic acid to identify and isolate aBDV-related human virus.

The B8 cDNA clone characterized in highly specific represents a highlyspecific nucleic acid probe for the BDV agent. The clone most likelyrepresents a cDNA copy of the 0.85 kb mRNA species. The fact that thisclone strongly hybridizes to two other small mRNAs (3.6 and 2.1 kb)suggests that these mRNAs share common sequences. They could represent anested set of partially overlapping mRNAs or mRNAs with common 5'leaderor 3'specific sequences. The antigenic cross reaction between the 38/39and 24 kD proteins suggests that the coding regions for the proteinsoverlap. The positive and negative strand probes hybridize with equalintensity to a 10.5 kb RNA species in BDV-infected rat brain RNA,strongly suggesting that the 10.5 kb RNA represents the viral genome.

The biology of the BDV agent resembles rabies virus in that both virusesare extremely neurotropic (Carbone, Virol. 61:3431 (1987)). Lymphocyticchoriomeningitis virus (LCM) and BDV have similar immunopathologicalmechanisms which contribute to disease (Richt, J. Exp. Meal. 170:1045(1989)). Both rabies virus and LCM viruses have negative strand RNAgenomes. However, the production of small cross-hybridizing mRNAs by theBDV agent is similar to the organization and gene expression observedfor coronaviruses (Holmes, in: Virology, 2nd Ed., eds. Fields and Knipe,vol I, pp. 841-856 (1990)). Thus, the isolation of cDNAs for the otherBDV mRNAs and the genomic BDV RNA will be required to resolve thequestion of the genetic organization of the BDV. The molecular clonedescribed in this report will allow further studies on the pathogenesisof Borna disease and allow the extension of such studies to humandisease. The BDV agent appears to represent a new family or subfamily ofviruses containing a unique genetic organization with highlyneurovirulent biological properties and the ability to cause behavioraldisease.

According to the present invention, DNA molecules are provided which arecomplementary to the Borna Disease Virus genome or the messenger RNA(mRNA) transcribed from viral sequences. Complementary sequences can bedetermined by inspection of sequences, adenine being complementary touracil or thymine, and cytosine being complementary to guanine.Alternatively, complementarity can be determined by hybridization;single stranded sequences which are complementary hybridize to formduplex molecules under stringent hybridization conditions, as are knownin the art.

Although applicants do not wish to be bound by any particular theory, itis believed that the BDV genome comprises RNA which is positive inpolarity. That is to say, the genome is of the same polarity as the mRNAspecies. The genome is presumably transcribed into an intermediate ofopposite polarity (negative) which is then transcribed into messageswhich are translated into proteins. The negative strand observed mayalso be a replicative intermediate. Alternatively, the genome itself maybe a double stranded RNA molecule.

Antibodies for immunoprecipitation of BDV proteins can be polyclonal ormonoclonal. Both are known in the art. (See for example, Haas, J. gen.Virol. 67:235 (1986). They can be raised against mixtures of viralproteins or purified proteins. They can be raised against syntheticpeptides or fusion proteins containing BDV epitopes, as are disclosedherein.

The preparations of BDV proteins according to the present invention, aresubstantially free of mammalian proteins. They can be synthesized invitro using techniques of synthetic chemistry, or translated usingnon-mammalian cell-free translation systems. Alternatively, they can beproduced in recombinant organisms, such as E. coli carrying cloned BDVgenes. The proteins can be fused to other non-BDV proteins, such as thebacterial β-galactosidase, as taught above.

Preparations which are substantially free of mammalian proteins compriseat least about 50% non-mammalian proteins and preferably greater thanabout 75% non-mammalian protein. More preferred are preparations whichhave less than about 10% or less than about 5% mammalian proteins.Preparations which are substantially free of mammalian proteins do notdemonstrate reactivity with rat or rabbit antisera in immunoblots toproteins other than the 38/39 kD, the 24 kD, the 15.5 kD, the 14.5 kD,and the 13 kD which are encoded by Borna Disease Virus. Due to themethods of producing the proteins which are provided herein, theproteins can be essentially free of mammalian proteins, i.e., containingless than about 0.05% mammalian proteins.

It is a finding of the present invention that mammalian cell cultureswhich have been infected with human cerebro-spinal fluid (CSF) ofpatients having behavioral diseases elaborate RNA species which aresimilar to, but different from, Borna Disease Virus-encoded RNA. Thehuman CSF-infected cells express RNA species which are of differentsizes than those of BDV-infected cells. Furthermore the intensity of thehybridization signal of the RNA species expressed by the humanCSF-infected cells is weaker than that of BDV-infected cells, whenhybridization is to BDV-specific nucleotide probes. These resultsdemonstrate that some humans having behavioral diseases, such asschizophrenia and bipolar depression, are infected with a BDV-relatedhuman virus. The ability of BDV to cause behavioral diseases in animals,suggests that the BDV-related human virus plays an etiological role inthe human psychiatric disorders.

The cross-hybridization of the BDV-specific nucleotide probes with thenucleic acids from the human CSF-infected cell lines, and thecross-reactivity of human antisera with persistently BDV-infected cells(Rott, Science 228:755 (1985)) demonstrates that the BDV-specificproteins and nucleic acids taught herein, as well as BDV-specificantibodies can be used to isolate the BDV-related human virus.Furthermore, the results disclosed here demonstrate that theBDV-specific proteins and nucleic acids can be used diagnostically inhumans as well as in other mammals.

In order to isolate the BDV-related human virus specific cDNA, themethods taught for isolation of BDV-specific cDNA are followed. However,instead of constructing the cDNA library from mRNA isolated fromBDV-infected MDCK cell lines, the cDNA library is constructed from humanCSF-infected cell lines, the human CSF coming from patients withbehavioral disorders. Preferably the human CSF donors are pre-screenedfor serum immunofluorescence, as taught by Rott. The clones are selectedeither with BDV-specific antibodies or with BDV-specific DNA probes, asprovided herein.

Immunological detection of BDV and BDV-related infections can be carriedout according to any techniques known in the art. These may involveradioimmunoassay, ELISA, Western blots, radioimmunoprecipitation assays,etc. The particular techniques are practiced as are known in the art.Use of the substantially purified preparations of BDV-encoded proteinspermits specific determination of the presence of a BDV or BDV-relatedhuman virus infection.

Kits are provided by the present invention for carrying outimmunologcial detection of BDV and BDV-related human virus. The kitscomprise a solid support with a preparation of Borna DiseaseVirus-encoded protein bound to the solid support. The solid support mayinclude polystyrene or other plastic substance to which proteins arereadily bound. The solid support can be in the form of a microtiter dishfor performing ELISA assays, or a dipstick for performing asandwich-type assay. Alternatively the solid support can be a matrixmaterial for packing a column, such as is used in immunoaffinitychromatography. Other reagents for carrying out the assay may also bepresent in the kit, such as enzymes and chromogenic substrates tofacilitate the detection of antibody-antigen complexes. The kits maycontain one or more of the purified BDV-encoded proteins.

Methods for determining the presence of a BDV or BDV-related human virusare also contemplated as within the scope of the invention which utilizenucleic acid hybridization techniques. These can be done as is known inthe art. Hybridizations can be performed on electrophoreticallyseparated RNA species isolated from cells, as in the Northern technique.Alternatively, hybridizations can be performed in situ on fixed cells.Other assay techniques which are known can be used employing as specificprobes the BDV-specific and BDV-related human virus-specific nucleicacids provided herein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 707 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Borna Disease Virus                                             (vii) IMMEDIATE SOURCE:                                                       (B) CLONE: B8                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       ATAAAAAACCAAATGCGGCAAACCCCCCGCGACCTGTGATGAGTTCCGACCTCCGGCTGA60                CATTGCTTGAATTAGTCAGGAGGCTCAATGGCAACGCGACCATCGAGTCTGGTCGACTCC120               CTGGAGGACGAAGAAGATCCCCAGACACTACGACGGGAACGGTCGGGGTCACCAAGACCA180               CGGAAGATCCCAAGGAATGCATTGACCCAACCGGTAGACCAGCTCCTGAAGGACCTCAGG240               AAGAACCCCTCCATGATCTCAGACCCAGACCAGCGAACCGGAAGGAGCAGCTATCGAATG300               ATGAGCTTATCAAGAAGCTAGTGACGGAGCTGGCCGAGAATAGCATGATCGAGGCTGAGG360               AGGTGCGGGGCACTCTTGGGGACATCTCGGCTCGCATCGAGGCAGGGTTTGAGTCCCTGT420               CCGCCCTCCAAGTGGAAACCATCCAGACACTCAGCGGTGCGACCACTCCGATAGCATCAG480               AATTCCTTGGCGAGAACATCAAGATACTGGATCGCTCCATGAAGACAATGATGGAGACAA540               TGAAGCTCATGATGGAGAAGGTGGACCTCCTCTACGCATCAACCGCCGTTGGGACCTCTG600               CACCCATGTTGCCCTCCCATCCTGCACCTCCGCGCATTTATCCCCAGCTCCCAAGTGCCC660               CGACAGCGGATGAGTGGGACATCATACCATAAAAAAAAAAAAAAAAA707                            __________________________________________________________________________

What is claimed is:
 1. A preparation of proteins encoded by the DNA ofSEQ ID NO:1, wherein the proteins are selected from the group consistingof an approximately 24 kD protein, an approximately 15.5 kD protein, anapproximately 14.5 kD protein, and an approximately 13 kD protein. 2.The preparation of claim 1 comprising less than about 0.5% mammalianproteins.
 3. A kit for detecting a Borna Disease Virus infection inanimals or a Borna Disease Virus-related human virus infection inhumans, comprising:a solid support; and a preparation of virus-encodedproteins substantially free of mammalian proteins, wherein saidvirus-encoded proteins are encoded by the DNA of SEQ ID NO:1 and whereinsaid virus-encoded proteins are bound to said solid support.
 4. The kitof claim 3 wherein the solid support is a microtiter dish.
 5. The kit ofclaim 3 wherein the solid support is a dipstick.
 6. The kit of claim 3wherein said virus-encoded protein is selected from the group consistingof: an approximately 24 kD protein, an approximately 15.5 kD protein, anapproximately 14.5 kD protein, and an approximately 13 kD protein.
 7. Apreparation of virus-encoded proteins substantially free of mammalianproteins, wherein said virus-encoded proteins are made by the process oftranscribing and translating a cDNA molecule having the sequence of SEQID NO:1, wherein the sequence encodes Borna Disease Virus proteins.
 8. Apreparation of virus-encoded proteins substantially free of mammalianproteins, wherein said virus-encoded proteins are made by the processof: polymerizing amino acids according to the amino acid sequencedetermined by the nucleic acid sequence of SEQ ID NO:1.
 9. Thepreparation of claim 7 wherein said transcribing and translating areperformed in a cell-free system.
 10. The preparation of claim 7 whereinsaid transcribing and translating are performed in a recombinantDNA-containing cell.